Cloning and expression of human GMP synthetase, its use in screening for inhibitors of human GMP synthetase and inhibitors of human GMP synthetase

ABSTRACT

One aspect of the present invention is a purified human GMP synthetase and a method of purifying it from naturally occurring sources. Another aspect of the present invention is a recombinant human GMP synthetase as well as DNA sequences coding for human GMP synthetase, expression vectors comprising such a coding sequence, and host cells transformed with these expression vectors capable of producing human GMP synthetase. Also forming part of this invention is a recombinant process for the production of GMP synthetase. Further provided is a method of purifying GMP synthetase from natural or recombinant sources. Other aspects of the invention include antibodies to human GMP synthetase and the use of such antibodies to assay for human GMP synthetase. Another aspect of this invention is the use of purified naturally occurring human GMP synthetase or recombinant human GMP synthetase to identify inhibitors of GMP synthetase activity. Another aspect of the invention is inhibitors of human GMP synthetase activity obtained using purified human GMP synthetase. Such inhibitors preferably have an IC 50  of 5 μM or less, more preferably 1 μM or less.

This is a continuation of Ser. No. 08/461,489, filed Jun. 5, 1995, nowabandoned which is a Divisional of application Ser. No. 08/224,917,filed Apr. 8, 1994, and now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to purified human GMP synthetase, its productionby recombinant technology and nucleic acid sequences encoding for humanGMP synthetase and its use in assaying for inhibitors of human GMPsynthetase activity and the inhibitors identifiable by such assays.

Human guanosine 5'-monophosphate (GMP) synthetase is a key enzyme in thede nova synthesis of guanine nucleotides. Biosynthesis of guaninenucleotides is not only essential for deoxyribonucleic acid (DNA) andribonucleic acid (RNA) synthesis, but also for providing guanosine5'-triphosphate (GTP) which is involved in a number of cellularprocesses important for cell division. GTP hydrolysis is required formicrotubule assembly, protein glycosylation, synthesis of adediinenucleotides, protein translation and activation of G proteins. A keystep in the de novo synthesis of guanine nucleotides is the conversionof inosine 5'-monophosphate (IMP) to guanosine 5'-monophosphate (GMP).Two enzymes are involved in converting IMP to GMP: IMP dehydrogenase (EC1.1.1.205) which catalyzes the oxidation of IMP to XMP (xanthine5'-monophosphate) and GMP synthetase (EC 6.3.5.2.) which catalyzes theamination of XMP to GTP. The reaction catalyzed by GMP synthetase is:

    XMP+ATP+glutamine→GMP+AMP+PP.sub.i +glutamate

Both GMP synthetase and IMP dehydrogenase exhibit elevated levels ofactivity in rapidly proliferating cells, such as neoplastic andregenerating tissues. Inhibition of GMP synthetase or IMP dehydrogenasehas been shown to result in the inhibition of cell growth. Because ofthis anti-proliferative effect of GMP synthetase and IMP dehydrogenaseinhibitors, both enzymes are potential targets for anti-cancer andimmunosuppressive therapies. In fact, in recent clinical trials, apotent inhibitor of IMP dehydrogenase, mycophenolic acid, was shown tobe effective in treatment of transplant rejection and rheumatoidarthritis.

The inhibition of lymphocyte proliferation by mycophenolic acid isclosely correlated with the lowering of the intracellular pool ofguanine nucleotides. Furthermore, this inhibition of proliferation canbe blocked by the addition of exogenous guanosine. These data suggestthat the immunosuppressive activity of mycophenolic acid is a result ofthe depletion of the guanine nucleotide pool. Since GMP synthetase iscrucial for the de novo synthesis of guanine nucleotides, the inhibitionof GMP synthetase should also result in the depletion of guaninenucleotides and therefore could be a target for immunosuppressive andcancer therapies.

Despite its significance, the information available for human GMPsynthetase is limited. Heretofore, the human enzyme has not beenobtained in a purified state. Even though a partially purifiedpreparation from human fibroblast cells (T. Page, B, Bakay and W. L.Nyhan; Int. J. Biochem., 16, 117-120, (1984)) has been reported, itcontained varying amounts 5'-nucleotidase, purine nucleosidephosphorylase, and GMP kinase. The degree of purification of the enzymewas approximately 50-fold over the naturally occurring material and itsspecific activity was reported to be 5990 pmoles guanine compoundproduced/min/mg. By contrast, the present invention discloses a purifiedhuman GMP synthetase purified 936-fold over the naturally occurringmaterial with a specific activity of at least 2,500,000 pmoles adenosine5'-monophosphate (AMP) produced/min/mg. GMP synthetases of mammalianorigin have been isolated in varying degrees of purity from a variety ofsources. However, mammalian GMP synthetases, even if available in a highdegree of purity, are of limited utility in screening for inhibitors forthe purposes of human therapy.

Genetic information for human GMP synthetase is not available. Althoughthe cDNA for GMP synthetase has been isolated from E. coli, B.subtilisand D. discoideum, no human or mammalian cDNAs or genes for GMPsynthetase are available. Therefore it has not been possible to producehuman GMP synthetase by recombinant DNA technology.

Accordingly, human GMP synthetase has not been available in a formsuitable either for elucidation of its mechanism or for its use inscreening for potential inhibitors. Neither has there been sequenceinformation such as, for example, cDNA sequences coding for human GMPsynthetase which would enable the production of human GMP synthetase byrecombinant DNA technology. For these reasons, it is desirable toproduce pure, well characterized human GMP synthetase in largequantities for the purposes, for example, of elucidating the mechanismsof its activity, identifying its role in various disease states andscreening for therapeutically significant inhibitors of guaninenucleotide biosynthesis. Furthermore, it would be desirable to producehuman GMP synthetase by means of recombinant DNA technology because itwould make the enzyme available in much larger amounts than is practicalin purification from natural sources.

SUMMARY OF RELATED ART

Human GMP Synthetase; T. Page, B, Bakay and W. L. Nyhan; Int. J.Biochem., 16, 117-120, (1984); disclosed GMP synthetase from humanfibroblasts, purified approximately 50-fold by ammonium sulfatefractionation and gel filtration.

Purification and Characterization of GMP Synthetase from Yoshida SarcomaAscites Cells; K. Harai, Y. Matsuda and H. Nakagawa; J. Biochem., 102,893-902, (1987); disclosed GMP synthetase purified from Yoshida Sarcomamouse ascites cells by means of procedures including centrifugalfractionation. The purified enzyme was shown to be homogeneous onSDS-polyacrylamide gel electrophoresis and isoelectric focusing inpolyacrylamide gel.

GMP Synthetase from Ehrlich Ascites Cells; T. Spector; MethodsEnzymology, 51, 219-224, (1978); described coupled spectrophotometricand radiochemical assays for measuring GMP synthetase. The formation ofAMP and/or GMP is coupled to the oxidation of NADH as mediated via AMPkinase and/or GMP kinase, pyruvate kinase, and lactate dehydrogenase.

Nucleotide Sequence of the guaa Gene Encoding GMP Synthetase ofEscherichia coli K12; A. A. Tiedeman, J. M. Smith and H. Zalkin; J.Biol. Chem. 260, 8676-8679, (1985); reported the cloning and DNAsequence of a 1.7-kilobase pair BanII-PvuII fragment containing the E.coli guaA structural gene. A single open reading frame of 1575nucleotides was found and sequenced. The deduced amino acid sequencecontained 525 residues having a calculated subunit M_(T) of 58,604.

Functional Cloning of a Dictyostelium discoideum cDNA Encoding GMPSynthetase; M. M. Van Lookeren Campagne, J. Franke and R. H. Kessin; J.Biol. Chem. 266, 16448-16452 (1991); disclosed a functional cloningprocedure to recover a cDNA coding for the GMP synthetase ofDictyostelium discoideum. The enzyme is encoded by a single gene. Theopen reading frame encodes a protein of 718 amino acids with a predictedmolecular mass of 79.6 kDa.

Cloning and Sequence of Bacillus subtilis purA and guaa, Involved in theConversion of IMP to AMP and GMP; P. Mantsala and H. Zalkin; J.Bacteriology 174, 1883-1890 (1992); disclosed the nucleotide sequencesof the Bacillus subtilis genes purA, encoding adenylosuccinatesynthetase, and guaa, coding for GMP synthetase. The sequences weredetermined from a series of gene fragments isolated by polymerase chainreaction amplification, library screening and plasmid rescue techniques.

Guanosine Monophosphate Synthetase from Ehrlich ascites cells: Multipleinhibition by pyrophosphate and nucleosides; T. Spector, T. E. Jones, T.A. Krenitsky and R. J. Harvey; Biochimica et Biophysica Acta 452,597-607 (1976); disclosed nucleoside inhibitors of GMP synthetase fromEhrlich ascites cells as well as the inhibition of the enzyme by itsreaction product, pyrophosphate.

SUMMARY OF THE INVENTION

One aspect of the present invention is a purified human GMP synthetaseand a method of purifying it from naturally occurring sources. Themethod comprises homogenizing host cells producing human GMP synthetase,isolating the cytosolic component, fractionating the cytosolic componentto its protein fraction and subjecting the protein component tochromatographic purification.

Another aspect of the present invention is a recombinant human GMPsynthetase as well as DNA sequences coding for human GMP synthetase,expression vectors comprising such a coding sequence, and host cellstransformed with these expression vectors capable of producing human GMPsynthetase. In one aspect, the invention includes a nucleic acidmolecule included in, or derived from, the sequence: (GMPS.6 (SEQ ID NO:1)), which is capable of encoding for human GMP synthetase.

Another aspect of the invention is sets of degenerate polynucleotideprimers corresponding to spaced amino acid sequences of human GMPsynthetase for use in selectively amplifying human derived nucleic acidsequences which code for GMP synthetase. Each set of primers containssubstantially all of the possible coding sequences corresponding to thespaced amino acid sequences of human GMP synthetase. That is, each setincludes at least one primer species that can effectively hybridize withthe coding sequence of the corresponding amino acid region.

Further aspects of this invention include polynucleotide probes capableof hybridizing to nucleic acid sequences contained within the nucleicacid sequence of human GMP synthetase as well as such polynucleotideprobes labeled with at least one reporter molecule. Also included withinthis invention are assays for DNA sequences encoding for human GMPsynthetase using such labeled or unlabeled polynucleotide probes.

Also forming part of this invention is a recombinant process for theproduction of GMP synthetase. This recombinant process involvesinserting a DNA sequence encoding for GMP synthetase into an expressionvector, transforming a suitable host with the vector and isolating therecombinant protein expressed by the vector.

Further provided is a method of purifying GMP synthetase from natural orrecombinant sources. This method comprises the steps of homogenizingcells expressing human GMP synthetase and isolating the cytosoliccomponent, fractionating the cytosolic component to its protein fractionand subjecting the protein fraction to chromatographic purification.

Other aspects of the invention include antibodies to human GMPsynthetase and the use of such antibodies to assay for human GMPsynthetase.

Another aspect of this invention is the use of purified naturallyoccurring human GMP synthetase or recombinant human GMP synthetase toidentify inhibitors of GMP synthetase activity. In this aspect an assayis carried out by a method comprising contacting a medium suspected ofcontaining one or more of such inhibitors with purified human GMPsynthetase and measuring the activity of the human GMP synthetase.

Another aspect of the invention is inhibitors of human GMP synthetaseactivity obtained using purified human GMP synthetase. Such inhibitorspreferably have an IC₅₀ of 5 μM or less, more preferably 1 μM or less,and find use in inhibiting human GMP synthetase activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Nucleotide and predicted amino acid sequences of human GMPsynthetase. Amino acid residues are represented in single-letter code.Lines and numbers in parentheses mark the position of sequencescorresponding to the tryptic peptides determined from the purifiednative enzyme. Boxes mark the position of sequences corresponding to theoligonucleotide primers. Nucleotide residue 1 represents the start siteof translation.

FIG. 2. Functional expression of GMPS.6 (SEQ ID NO: 1) in a GMPsynthetase deficient strain of E. coli. The expression vectors NF.6 andES.6 were transfected into AT2465 E.coli, and the cultures were spreadon minimal media plates with or without guanosine as described inExample 2. Colony formation was monitored after 24 hours. To determineGMP synthetase activity, single colonies were selected, propagated andinduced with IPTG, as described in Example 2. Single colonies wereselected from "minus guanosine" plates with NF.6 transfected cells andfrom "plus guanosine" plates with ES.6 transfected cells. Singlecolonies of cells that were not transfected were selected from LBplates. Cultures were harvested and the cytosolic fractions were assayedfor GMP synthetase activity as described in Example 2.

FIG. 3. Purification of GMP synthetase from A3.01 cells. Samples fromeach step of the purification were subjected to SDS polyacrylamide gelelectrophoresis and stained with Coomassie blue. The purification stepand amount of protein loaded in each lane are as follows: lanes 1.35-60% ammonium sulfate precipitate, 6 μg; 2. DEAE-cellulose pool, 2.5μg; 3. Phenyl-5PW pool, 3 μg; 4. Mono Q pool, 3 μg.

FIG. 4. Restriction map of GMPS.6 (SEQ ID NO: 1) and probes. The regionof hatched bars indicates the position of the longest open readingframe. The scale gives length in base pairs. The black bar marks thelength and position of the PCR fragment pcr.2S8A (SEQ ID NO: 2). Theopen bars mark the length and position of the CDNA fragments used asprobes in northern and Southern hybridization analysis. The small arrowsmark the position and direction of oligonucleotide primers 2S and 8A inthe 5' to 3' orientation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes purified naturally occurring andrecombinant human GMP synthetase as well as methods of purifying humanGMP synthetase from cells expressing human GMP synthetase. Furtherincluded are recombinant processes for the production of human GMPsynthetase. The invention also includes nucleic acid sequences codingfor human GMP synthetase, expression vectors comprising this codingsequence and host cells transformed with these expression vectorscapable of expressing human GMP synthetase. Also included arepolynucleotide probes capable of hybridizing to nucleic acid sequencesof human GMP synthetase and assays for human GMP synthetase using suchprobes. Also included are antibodies to human GMP synthetase, assays forGMP synthetase using such antibodies and assays for inhibitors of humanGMP synthetase activity using the recombinant or purified natural enzymedisclosed by this invention. The invention also includes inhibitors ofhuman GMP synthetase preferably having an IC₅₀ of 5 μM or less.

Before proceeding further with the description of the specificembodiments of the invention a number of terms will be defined.

The term "purified human GMP synthetase" refers to a human GMPsynthetase which is essentially free, i.e. contains less than about 30%,preferably less than about 10%, and even more preferably less than about95% of the proteins with which the human GMP synthetase is naturallyassociated. Techniques for assessing purity are well known to the artand include, for example, sodium dodecyl sulfate (SDS) gelelectrophoresis, high pressure liquid chromatography (HPLC), isoelectricfocusing and capillary gel electrophoresis. Thus, the term purified alsorefers to a human GMP synthetase which appears as essentially one bandon SDS gel electrophoresis when visualized by Coomassie blue staining.Purity can alternatively be assessed by measurement of the activity ofthe human GMP synthetase, for example, by the rate at which the purifiedhuman GMP synthetase produces adenosine 5'-monophosphate (AMP). Thus,the term purified may also refer to a human GMP synthetase that producesμMP at a rate at least 100 fold faster than, preferably 500 fold fasterthan, and more preferably 900 fold faster than, the human GMP synthetasein its naturally occurring state when measured under the conditionsdisclosed herein. The term purified can also refer to a human GMPsynthetase that has a specific activity of at least 2.5 μmoles(2,500,000 pmoles) of AMP produced/min/mg when enzyme activity ismeasured using the conditions described in Example 1. The activity ofthe human GMP synthetase is measured by determining the rate at which itcatalyzes the conversion of XMP to GMP or ATP to AMP under theconditions described in the Examples 1 and 3.

The term "IC₅₀ " refers to the concentration of a compound that isrequired to inhibit by 50% the activity of purified human GMP synthetasewhen enzyme activity is measured under the conditions of the procedure#2, the radioactive assay, described in Example 5. Briefly, the assaymeasures the conversion of XMP to GMP or ATP to AMP in the presence orabsence of a suspected inhibitory compound. Therefore, the concentrationof the inhibitory compound in the assay that reduces the amount of GMPor AMP produced by 50% compared to the amount of GMP or AMP produced inthe absence of the compound is the IC₅₀ of that compound.

The term "polynucleotide" as used herein refers to a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides. This term refers only to the primary structure ofthe molecule. Thus, this term includes double and single stranded DNA,as well as double and single stranded RNA. It also includes modified,for example, by methylation and/or by capping, and unmodified forms ofthe polynucleotide.

The term "recombinant polynucleotide" as used herein refers to apolynucleotide of genomic, cDNA, semisynthetic or synthetic originwhich, by virtue of its origin or manipulation: (1) is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature and/or (2) is linked to a polynucleotide other than that towhich it is linked in nature.

The term "cDNA" or complementary DNA refers to single stranded or doublestranded DNA sequences obtained by reverse transcription of messengerRNA isolated from a donor cell. For example, treatment of messenger RNAwith a reverse transcriptase such as AMV reverse transcriptase or M-MuLVreverse transcriptase in the presence of an oligonucleotide primer willfurnish an RNA-DNA duplex which can be treated with RNase H, DNApolymerase and DNA ligase to generate double stranded cDNA. If desired,the double stranded cDNA can be denatured by conventional techniquessuch as shearing to generate single stranded cDNA.

An "expression vector" is any genetic element, e.g., a plasmid, achromosome, a virus, behaving either as an autonomous unit ofpolynucleotide replication within a cell (i.e. capable of replicationunder its own control) or being rendered capable of replication byinsertion into a host cell chromosome, having attached to it anotherpolynucleotide segment, so as to bring about the replication and/orexpression of the attached segment. Suitable vectors include, but arenot limited to, plasmids, bacteriophages and cosmids. Vectors willcontain polynucleotide sequences which are necessary to effect ligationor insertion of the vector into a desired host cell and to effect theexpression of the attached segment. Such sequences differ depending onthe host organism; they include promoter sequences to effecttranscription, enhancer sequences to increase transcription, ribosomalbinding site sequences and transcription and translation terminationsequences.

The term "transfer vector" refers to a plasmid that enables theintegration of a recombinant gene into virus DNA by homologousrecombination.

The term "host cell" generally refers to prokaryotic or eukaryoticorganisms and includes any transformable organism which is capable ofexpressing a protein and can be, or has been, used as a recipient forexpression vectors or other transfer DNA.

The term "transformed" refers to any known method for the insertion offoreign DNA sequences into a host cell whereby the inserted DNA isrendered capable of replication in the host cell. The transformationprocedure depends on the host cell being transformed. It can includepackaging the polynucleotide in a virus as well as direct uptake of thepolynucleotide. Transformation can result in incorporation of theinserted DNA into the genome of the host cell or the maintenance of theinserted DNA within the host cell in plasmid form. Methods oftransformation are well known in the art and include, but are notlimited to, viral infection, electroporation, lipofection and calciumphosphate mediated direct uptake.

It is to be understood that this invention is intended to include otherforms of expression vectors, host cells and transformation techniqueswhich serve equivalent functions and which become known to the arthereto.

The term "CDNA library" refers to a collection of host cells transformedwith CDNA sequences derived from a donor cell.

The term "reporter molecule" refers to a chemical entity capable ofbeing detected by a suitable detection means, including, but not limitedto, spectrophotometric, immunochemical, or radiochemical means.

The term a polynucleotide "derived from" a designated sequence, forexample, the GMPS.6 sequence depicted in FIG. 1, which includes the cDNAof human GMP synthetase, refers to a polynucleotide sequence which iscomprised of a sequence of approximately at least 6 nucleotides,preferably at least 8 nucleotides, more preferably at least 10-12nucleotides, and, even more preferably, at least 15-20 nucleotidescorresponding to, i.e., homologous or complementary to, a region of thedesignated sequence. The derived polynucleotide sequence is notnecessarily physically derived from the nucleotide sequence shown, butmay be derived in any manner, including for example, chemical synthesisor DNA replication or reverse transcription, which are based on theinformation provided by the sequences of bases in the region(s) fromwhich the polynucleotide is derived.

The term "inhibitor" refers to a compound or extract which decreases theenzyme activity of human GMP synthetase when enzyme activity is measuredin the presence of the inhibitor relative to the enzyme activitymeasured in the absence of the inhibitor. Specific methods of measuringenzyme activity in the presence of suspected inhibitors are describedmore fully in Example 5.

Purification Of Human GMP Synthetase

The general method of purifying human GMP synthetase by the method ofthis invention comprises the steps of cell lysis, homogenization,centrifugation, anion exchange chromatography, hydrophobic interactionchromatography, and high resolution anion exchange chromatography.

Typically, a human cell line such as, for example, a T-lymphoblastomacell line, a Jurkat cell line, or a B cell line expressing GMPsynthetase is cultured under conditions appropriate to the particularcell line being used in an appropriate aqueous medium. The invention isnot restricted to any particular types of human cell lines althoughtransformed cell lines may provide better results than tissue. Themedium will usually be a salt medium such as normal saline, phosphatebuffer, or phosphate buffered saline containing additives well known tothose skilled in the art and commercially available from a number ofsuppliers such as GIBCO and Hyclone. The cultured cells are thenhomogenized under conventional conditions such as, for example,sonication, freeze-thawing, and hypotonic lysis, and the cytosolicfraction is obtained by centrifugation and recovery of the supernatant.Differential salt fractionation, using, for example, ammonium sulfate,is employed to separate the GMP synthetase-rich protein fraction. Saltis then removed by one of a variety of known methods such as gelfiltration, dialysis and the like. The desalted protein fraction is thensubjected to anion exchange chromatography. This is preferably doneusing DEAE-cellulose or Q-Sepharose as examples of anion exchangematerial. Fractions showing GMP-synthetase activity are furthersubjected to hydrophobic interaction chromatography and a secondpurification via anion exchange chromatography. (GMP synthetase activityis measured by the spectrophotometric-coupled assay described by Spector(T. Spector; Methods Enzymology, 51, 219-224, (1978)). The assaymeasures the rate of production of AMP by GMP synthetase by coupling itto the oxidation of NADH via the auxiliary enzymes AMP kinase, pyruvatekinase and lactate dehydrogenase. In this fashion a purified human GMPsynthetase is obtained. It has a molecular weight of approximately 75kDa and is a single band on SDS gel electrophoresis when visualized byCoomassie blue staining. Furthermore, it has a specific activity of 2.5μmoles AMP produced/min/mg or greater, often in the range 2.5 to 4.0μmoles AMP produced/min/mg, in the spectrophotometric-coupled Spectorassay referenced above.

Recombinant Human GMP Synthetase

As described above, the present invention provides purified human GMPsynthetase. Purified human GMP synthetase may be used to design DNAprobes for obtaining genomic or CDNA clones for use in the recombinantproduction of human GMP synthetase. For example, the amino acid sequenceof purified protein can be determined by chemical techniques andoligonucleotide sequences coding for specific amino acid segments can bederived. Alternatively, the purified protein can first be subjected toenzymatic digestion using proteases such as trypsin, pepsin and the liketo obtain peptide fragments whose amino acid sequences can be determinedby techniques such as Edman degradation.

These amino acid sequences are used to construct oligonucleotide primersof complementary sequences which may be used in a polynucleotideamplification technique such as the polymerase chain reaction (PCR) (R.K. Saiki, D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T.Horn, K. B. Mullis, and H. A. Ehrlich, Science, 239, 487-491 (1989) toamplify cDNA from a human cell line to obtain a DNA sequence includedwithin the gene coding for human GMP synthetase. Due to the degeneracyof the genetic code, oligonucleotide primers of complementary sequencesare customarily prepared in all possible variations. On occasion,variation of the primer sequences may be limited based on homology withrelated enzymes and/or species specific codon usage. The identity of theDNA sequence obtained via amplification can be verified by subcloninginto a second vector and sequencing positive clones to establish thepresence of sequences corresponding to the originally usedoligonucleotide primers. This DNA sequence obtained as above can then beused to screen a cDNA library obtained from a human cell line expressingGMP synthetase to identify the complete nucleic acid sequence coding forthe enzyme.

The basic molecular biology techniques employed in accomplishingfeatures of this invention, such as RNA and DNA and plasmid isolation,restriction enzyme digestion, preparation and probing of a cDNA library,sequencing clones, constructing expression vectors, transforming cells,maintaining and growing cell cultures and other general techniques arewell known in the art, and descriptions of such techniques can be foundin general laboratory manuals such as Molecular Cloning: A LaboratoryManual by J. Sambrook, E. F. Fritsch and T. Maniatis, published by theCold Spring Harbor Laboratory Press, 2nd edition, 1989.

The DNA sequences contemplated by this invention compriseoligodeoxynucleotides coding for human GMP synthetase as well asfragments thereof, as well as all equivalent nucleotide sequences codingfor molecules with substantially the same human biological activity ashuman GMP synthetase. In addition to the polymerase chain reactiondescribed herein, preparation of such DNA sequences is also possible byother amplification techniques as well as known synthetic procedures.

A DNA sequence coding for human GMP synthetase can be cloned into avariety of expression vectors. The expression vectors include, forexample, plasmids, bacteriophages and cosmids. The particular vectorchosen should be compatible with the contemplated host cell, whether abacterium such as, for example, E. coli, or yeast or other cell. Aplasmid should have the proper origin of replication for the particularhost cell to be employed and suitable restriction sites that allow theligation of foreign DNA sequences. Furthermore, the plasmid shouldimpart to the transformed cell a phenotypic property that will enablethe transformed cells expressing the enzyme to be readily identifiedfrom cells that do not undergo transformation. Similar considerationsapply to nonplasmid vectors. Numerous expression vectors with a varietyof properties are available from commercial suppliers.

The DNA encoding the desired human GMP synthetase, whether in fused ormature form, and whether or not containing a signal sequence to permitsecretion, is ligated into an expression vector suitable for anyconvenient host. Both eukaryotic and prokaryotic host cells arepresently used in forming recombinant proteins. By way of illustrationand not limitation, eukaryotic systems include human fibroblast HeLacells and Jurkat T-cells and prokaryotic systems include E. coli.

The expressed enzyme is then isolated from lysed cells or from culturemedium and purified to the extent needed. Purification is carried out bymethods known to the art, including, but not limited to, saltfractionation, ion exchange chromatography, hydrophobic chromatography,gel filtration chromatography, affinity chromatography, isoelectricfocusing and centrifugation.

The recombinant human GMP synthetase contemplated by this inventionincludes all functionally equivalent enzymes obtained by expressing in ahost cell a DNA sequence encoding for human GMP synthetase or a fragmentthereof, as well as functionally equivalent sequences coding for humanbiologically active GMP synthetase.

In a preferred embodiment of this invention as described in more detailin Example 4, a DNA coding sequence for human GMP synthetase issubcloned into a baculovirus transfer vector. Insect cells areco-transfected with the transfer plasmid and baculovirus virion DNA. Afew days later, when polyhedra are evident in the culture, the culturefluid is harvested and several clones of the recombinant virus plaqueare purified. Expression levels of GMP synthetase of each of therecombinant viruses are determined by infecting insect cells andperforming enzyme assays on the lysates of the infected cells. One ofthe clones with a high level of expression is selected for large scaleproduction of the enzyme, wherein insect cells growing in serum freeculture fluid are infected with the recombinant baculovirus obtainedabove. Sometime later the cells are harvested by centrifugation. Thecells are suspended in hypotonic buffer and mechanically disrupted.Following clarification by centrifugation, the soluble proteins arefractionated by ammonium sulfate precipitation. The GMP synthetasecontaining fraction is desalted and the GMP synthetase is purified bysequential anion-exchange and hydrophobic interaction chromatography. Afinal purification using high resolution anion-exchange chromatographyresults in a purified preparation of GMP synthetase. The purifiedrecombinant human GMP synthetase is a single band on SDS gelelectrophoresis as visualized by Coomassie blue staining and has aspecific activity of 3.0 μmol AMP produced/min/mg or greater, often inthe range 3.0 to 4.4 μmol AMP produced/min/mg, when measured by thespectrophotometric-coupled Spector assay referenced earlier.

Assays For Inhibitors Of Human GMP Synthetase

The present invention also relates to assays using purified human GMPsynthetase to screen for and identify inhibitors of human GMPsynthetase. The assay is a method comprising contacting a mediumcontaining the suspected inhibitor with purified human GMP synthetaseand measuring the activity of the human GMP synthetase. The medium canbe an extract of biological origin, such as, for example, a plant,animal, or microbial cell extract, or alternatively, it may be asubstantially pure compound of synthetic origin, or a mixture ofcompounds thereof.

Enzyme activity can be measured by methods such as direct measurement ofthe products of the enzymatic reaction or by coupling the formation ofthe reaction product to the reaction of other detectable species. Forexample, in one method for determining enzyme activity the formation ofAMP and/or GMP is measured by coupling their formation to the oxidationof NADH as mediated via AMP kinase and/or GMP kinase, pyruvate kinase,and lactate dehydrogenase. The oxidation can be monitoredspectrophotometrically at 340 nm. In another method for measuring theenzyme activity, radiolabeled XMP or ATP is used as substrate and theformation of radioactive GMP or AMP is measured directly afterseparation of substrates and products by chromatographic methods such asthin layer chromatography or high pressure liquid chromatography.

Compounds are identified based on their ability to inhibit the activityof the purified human GMP synthetase disclosed herein. The assay is usedto identify compounds having an IC₅₀ of less than 25 μM, preferably,less than 20 μM, more preferably, less than 10 μM. The preferredinhibitors have an IC₅₀ greater than 0 and less than 5 μM. Usually, thepresent inhibitors have an IC₅₀ of less than 5 μM, preferably less than1 μM, generally 0.01 to 5 μM, more preferably 0.1 to 1 μM. Theinhibitors are used in a method for inhibiting human GMP synthetaseactivity by administering an effective amount of the inhibitor having anIC₅₀ of 5 μM or less. The effective amount is generally that amount thatresults in greater than 50% decrease in human GMP synthetase activity,preferably, greater than 70% decrease in human GMP synthetase activity,more preferably, greater than 90% decrease in human GMP synthetaseactivity.

Antibodies Against Human GMP Synthetase

The present invention also relates to antibodies, both polyclonal andmonoclonal, against human GMP synthetase. Methods of preparingpolyclonal antibodies are well known to the art. For example, animmunogenic conjugate comprising the human GMP synthetase or a fragmentthereof, optionally linked if necessary to a carrier protein, is used toimmunize a selected mammal (e.g. mouse, rabbit, goat etc.). Serum fromthe immunized mammal is collected and treated according to knownprocedures to separate the immunoglobulin fraction. In one approach inthe present invention, the human GMP synthetase is expressed as a fusiontransfer protein, such as, for example, glutathione transferase fusionprotein, which is used to immunize rabbits according to a conventionalprocedure.

Monoclonal antibodies are prepared by standard hybridoma cell technologybased on that reported by Kohler and Milstein in Nature 256 (1975)495-497. Briefly, spleen cells are obtained from a host animal immunizedwith human GMP synthetase protein or a fragment thereof, optionallylinked to a carrier if necessary. Hybrid cells are formed by fusingthese spleen cells with an appropriate myeloma cell line and cultured.The antibodies produced by the hybrid cells are screened for theirability to bind to purified human GMP synthetase. A number of screeningtechniques well known in the art, such as, for example, forward orreverse enzyme-linked immunosorbent assay (ELISA) screening methods maybe employed. The hybrid cells producing such antibodies are thensubjected to recloning and high dilution conditions in order to select ahybrid cell that secretes a homogeneous population of antibodiesspecific to human GMP synthetase. The antibodies of this invention finduse in the detection of human GMP synthetase.

The antibodies of this invention can be conjugated to a reportermolecule by techniques well known in the art. Typically the reportermolecule contains a functional group suitable for attachment of thereporter group to the antibody. The functional groups suitable forattaching the reporter group are usually activated esters or alkylatingagents which react with nucleophilic groups such as, for example, amino,hydroxyl or thiol groups on the antibody. Details of these and relatedtechniques for attaching reporter groups to proteins are described inreviews such as, for example, Enzyme-Immunoassay by E. T. Maggio,published by the CRC Press, Boca Raton, Fla., 1980.

Generation of Nucleic Acid Probe

Purified human GMP synthetase is subjected to peptide digestion, using,for example, trypsin, pepsin, or other proteases and the like, and thepeptides are resolved by chromatographic techniques such as, forexample, reverse phase high pressure liquid chromatography. The peptidesare sequenced. Degenerate oligonucleotides are synthesized correspondingto the derived amino acid sequences of the above peptides.Oligonucleotides are synthesized in both the sense and antisensedirections for each peptide and are used as primers in an amplificationby the polymerase chain reaction. Since the location of the peptides inthe protein sequence is unknown, each oligonucleotide is used withanother as primers. A template for the PCR reaction is constructed bygenerating the cDNA complementary to RNA, such as polyadenylated RNA,from a human cell line expressing GMP synthetase. This is accomplishedby extracting the total cellular RNA from the cell line of interest,separating the RNA fraction of interest by column chromatography usingthe appropriate column substrate, such as, for example, in the case ofpolyadenylated RNA, an oligo-dT cellulose column, and treating it withreverse transcriptase. A DNA amplification reaction is carried out byrepeated primer initiated strand extension and fragments are generatedand isolated by appropriate techniques. Polynucleotide probes areprepared by conjugation of reporter molecules to fragments isolated asabove.

Identification of DNA Sequence Encoding For Human GMP Synthetase

The fragments obtained above are used to screen a cDNA library preparedby using RNA from human cells with a suitable cDNA cloning vector. Awide variety of cloning vectors are available, including lambda vectorsand phagemid vectors. Second generation "Okayama-Berg" vectors are alsoavailable for efficient cDNA cloning and subsequent expression inmammalian systems. In one such instance using polyadenylated RNA, anumber of positive clones are isolated of which the four longest areoverlapping. The longest sequence, GMPS.6 (SEQ ID NO: 1), is shown inFIG. 1 and was approximately 2.2 kb. In FIG. 1, amino acid residues arerepresented in single-letter code. Lines and numbers in parentheses markthe position of sequences corresponding to the tryptic peptidesdetermined from the purified native enzyme. Boxes mark the position ofsequences corresponding to the oligonucleotide primers. Nucleotideresidue 1 represents the start site of translation.

Sequence analysis of GMPS.6 (SEQ ID NO: 1) reveals an open reading frameof 2079 base pairs (FIG. 1). This open reading frame yields a protein of693 amino acid residues and a predicted molecular weight of 76,725. Thispredicted size is in good agreement with the 75 kDA size of the purifiedhuman GMP synthetase from natural sources obtained above. The sequencesof all nine tryptic peptides are contained within the predicted proteinsequence and are shown in Table 2 in Example 2.

Expression Of Recombinant Human GMP Synthetase

One embodiment of the invention is exemplified by a recombinant proteinprepared by subcloning the cDNA sequence spanning bases 1 to 2090 ofGMPS.6 (SEQ ID NO: 1) into the NcoI/Pst I cloning site of theprokaryotic expression vector, pTRC.99A (Pharmacia) which contains anampicillin resistance marker and the trc promoter that is inducible byIPTG. This plasmid is designated NF.6 (NF designates "no fusion") andcontains the entire open reading frame and all of the 3'-untranslatedsequence, but none of the 5'-untranslated sequence. A host cell istransfected with the NF.6 plasmid and cultured to express therecombinant protein. Purification of the recombinant protein yields aproduct of greater than 95% of purity as determined by SDS gelelectrophoresis and Coomassie blue staining. The recombinant enzyme ofthis embodiment has the identical kinetic and biochemical properties asthe purified human enzyme described previously.

Functional expression of human GMP synthetase in this fashion is furtherverified by testing for complementation of guanosine requirement inAT2465 E.coli transfected cells which do not grow in absence ofguanosine. A second expression vector is constructed as described aboveexcept that two stop codons are inserted into the ligatedoligonucleotide 39 base pairs downstream from the start site of thetranslation. This expression vector is designated ES.6 (ES abbreviates"early stop") and it coded for a truncated protein, as expected. BothES.6 and NF.6 are transfected into AT2465 E. coli, and the cells areallowed to grow on minimal medium plates containing ampicillin and IPTGwith or without guanosine. As shown in FIG. 2, cells that are nottransfected do not produce colonies on either plate due to the absenceof ampicillin resistance in the parental AT2465 cells. Cells that aretransfected with either NF.6 or ES.6 produce colonies in the presence ofguanosine. Only cells transfected with NF.6 form colonies in the absenceof guanosine. This result shows that GMPS.6 (SEQ ID NO: 1) contains acDNA that can complement a host E. coli that was deficient in GMPsynthetase. When this cDNA is mutated in ES.6 by the insertion of stopcodons, it can no longer complement growth in the absence of guanosine.These results show complementation by the transfected human DNA.

The transfected cells also show the presence of GMP synthetase enzymeactivity. Single colonies are selected from plates without guanosine(NF.6 transfected cells) or with guanosine (ES.6 transfected cells). Asshown in FIG. 2, GMP synthetase activity is present in NF.6 transfectedcells and absent in ES.6 transfected cells. Furthermore, the activity isinduced by the presence of IPTG. There is no detectable GMP synthetaseactivity in untransfected cells. These findings confirm thecomplementation results and demonstrate the cloning of a cDNA encoding afunctional human GMP synthetase.

EXAMPLES

Materials

Restriction endonucleases and all enzymes used for plasmid constructionwere purchased from New England Biolabs (Beverly, Mass.); oligo(dT)-cellulose from Pharmacia LKB (Uppsala, Sweden); Taq DNA polymerasefrom Perkin Elmer Cetus (Norwalk,Conn.); [α-³² P]dCTP from Amersham(Arlington Heights, Ill.); [α-³² P]UTP from Du Pont NEN (Boston, Mass.).All other chemicals were purchased from Sigma (St. Louis, Mo.).

Cells and Media

A3.01 cells were obtained from Dr. Thomas Folks (National Institute ofHealth, Bethesda, Md.. Folks, T., Benn, S., Rabson, A., Theodore, T.,Hoggan, M. D., Martin, M., Lightfoote, M., and Sell, K. (1985) Proc.Natl. Acad. Sci. USA 82, 4539-4543). The A3.01 cells were cultured inRPMI 1640 media containing 5% fetal bovine serum and 10 μg/ml gentamicin(HyClone, Logan, Utah). CEM, HL60, U937 and WI38 cells were purchasedfrom American Type Culture Collection (Rockville, Md.); Jurkat and UCcells were from Dr. Jeffrey Northrop (Stanford University MedicalCenter, Stanford, Calif.); VB and HFF cells were provided by the cellculture facility at Syntex Corporation (Palo Alto, Calif.). Normalperipheral T lymphocytes were isolated from a healthy donor according toan approved protocol and purified as described by Eugui, E. M., andAlmquist, S. J. (1990) Proc. Natl. Acad. Sci. USA 87, 1305-1309.

The bacterial hosts, SURE and XL1-Blue, used in library amplificationand screening, were provided by Stratagene (La Jolla, Calif.). TheAT2465 strain of E. coli was obtained from the E. Coli Genetic StockCenter (Yale University, New Haven, Conn.). The AT2465 cells have thefollowing chromosomal markers: thi-1, guaA21, relA1, λ- and spoT1.AT2465 cells do not grow in minimal media in the absence of guanosineand thiamin (Taylor, A. L., and Trotter, C. D. (1967) Bacteriol. Rev.31, 332-353 and references therein).

Example 1

Purification of Naturally Occurring Human GMP Synthetase

A particularly preferred method of purification of naturally occurringhuman GMP synthetase from A3.01 cells is described below.

Step 1: Homogenization. In a typical purification, A3.01 cells werecultured in RAMI 1640 media containing 5% fetal bovine serum and 10μg/ml gentamicin (Hyclone, Logan, Utah). The cells were lysed in 15 mlof buffer A (20 mM Tris.HCl, pH 7.6, 0.1 mM DTT, 0.5 mM EDTA, 10%glycerol) by the use of a glass teflon homogenizer. The homogenate wascentrifuged at 15,000×g for 20 min and the pellet was discarded.Purification was carried out with the supernatant (cytosol).

Step 2: Ammonium Sulfate Fractionation. Ammonium sulfate was added tothe cytosol fraction obtained above until 35% saturation and theprecipitated proteins were removed by centrifugation at 20,000×g for 20min and discarded. The proteins in the supernatant were precipitatedwith further addition of ammonium sulfate (60% saturation). Theseprecipitated proteins, which include GMP synthetase, were recovered bycentrifugation as above. The protein pellet was dissolved in 12 mlbuffer A. Ammonium sulfate was removed from the protein solution by gelfiltration chromatography on PD-10 columns (Pharmacia).

Step 3: DEAE-Cellulose Chromatography. The desalted protein fractionobtained above was applied to a 1.5×7.3 cm column of DEAE-cellulose(Whatman) equilibrated in buffer A. The enzyme was eluted with a 60-mlgradient of 0 to 0.5M sodium chloride in buffer A. The fractionscontaining GMP synthetase activity were pooled.

Step 4: Phenyl-5PW Chromatography. The DEAE cellulose pool obtainedabove was diluted with an equal volume of 0.2M potassium phosphate, pH7.0. 1.7M ammonium sulfate, 20% glycerol. The protein sample was appliedto a 8 mm×7.5 cm column of Phenyl-5PW (TosoHaas, Montgomeryville, Pa.)equilibrated in buffer B (0.1M potassium phosphate, pH 7.0, 1.7Mammonium sulfate, 10% glycerol). GMP synthetase was eluted with a 40-mlgradient of 1.7 to 0M ammonium sulfate in buffer B and the fractionscontaining active enzyme were pooled.

Step 5: Mono-Q Chromatography. The Phenyl-5PW pool obtained above wasdesalted as above and applied to a Mono Q HR 5/5 column (Pharmacia)equilibrated with buffer C (20 mM Tris. HCl, pH 8.0, 0.1 mM DTT, 20%glycerol). GMP synthetase was eluted with a 30-ml gradient of 0-0.3Msodium chloride in buffer C. The GMP synthetase fractions that containeda single Coomassie blue-staining band at 75 kDa were pooled. Thismaterial is purified human GMP synthetase.

A purification table from a representative purification for the varioussteps of the process is shown in Table 1 below. Enzyme activity wasdetermined by using the spectrophotometric-coupled assay of Spectorperformed exactly as described (T. Spector; Methods Enzymology, 51,219-224, (1978)), incorporated herein by reference. This assay measuredthe rate of AMP production by GMP synthetase, and the level of AMP ismeasured through the activity of three auxiliary enzymes. When crudeenzyme samples such as those in steps 1 to 3 of the purification wereexamined, this assay method measured a high rate of AMP formation evenin the absence of the GMP synthetase substrate, XMP. This rate isreferred to as background rate. The GMP synthetase activity shown inTable 1 below was the rate of AMP formation in the presence of all thesubstrates subtracted by the background rate when XMP is omitted. TheGMP synthetase activity determined by this assay was the same as theactivity determined by measuring radioactive GMP formation as describedbelow in Example 3.

                                      TABLE 1                                     __________________________________________________________________________              total protein                                                                       total activity                                                                        specific activity                                       sample (mg) (μmol AMP/min) (μmol AMP/min - mg) fold purified          __________________________________________________________________________    cytosol   2478.55                                                                             7.42    0.0030     1                                            35-60% AS precipitate 1095.46 10.24 0.0094 3                                  DEAE-cellulose pool 46.64 3.25 0.0670 23                                      Phenyl-5PW pool 3.95 1.59 0.4038 135                                          Mono Q pool 0.17 0.47 3.0 936                                               __________________________________________________________________________

FIG. 3 shows the SDS polyacrylamide gel electrophoresis of each step inthe purification. Samples from each step of the purification weresubjected to SDS polyacrylamide gel electrophoresis and stained withCoomassie blue. The purification step and amount of protein loaded ineach lane are as follows: lanes 1. 35-60% ammonium sulfate precipitate,6 μg; 2. DEAE-cellulose pool, 2.5 μg; 3. Phenyl-5PW pool, 3 μg; 4. MonoQ pool, 3 μg. The purified human GMP synthetase was essentially one bandafter the final purification step. The above purification was repeatedon several occasions and samples of human GMP synthetase with specificactivities in the range of 2.5-4.0 μmol AMP produced/min/mg wereroutinely obtained.

Example 2

Generation of Nucleic Acid Probes and Identification of the Gene forHuman GMP Synthetase

GMP synthetase from A3.01 cells was purified to homogeneity as describedabove and digested with trypsin. Nine tryptic peptides were resolved byreverse-phase high-pressure liquid chromatography (RP-HPLC) andsubjected to sequence analysis by Edman degradation using an ABI470Asequencer (Applied Biosystems, Foster City, Calif.). The sequences ofthe nine peptides are shown in Table 2 below. The sequences arerepresented by the standard one-letter code. The letter "X" indicatesthat no amino acid can be assigned from the corresponding cycle of Edmandegradation.

                  TABLE 2                                                         ______________________________________                                        Peptide   Sequence                                                            ______________________________________                                        1         NIVAGIANESK (SEQ ID NO: 3)                                            2 ALNQEQVIAVXIDNGFM (SEQ ID NO: 4)                                            3 VINAAHSFYNGTXXLPISDED (SEQ ID NO: 5)                                        4 IIGDTFV (SEQ ID NO: 6)                                                      5 ANEVIGXMNLK (SEQ ID NO: 7)                                                  6 VIEPLKDFIKDEVR (SEQ ID NO: 8)                                               7 PFPGPGLAI (SEQ ID NO: 9)                                                    8 FITSDFMTGIPATPGNEIXV (SEQ ID NO: 10)                                        9 IMYDLTSKPPGTTE (SEQ ID NO: 11)                                            ______________________________________                                    

Based on the peptide sequences, degenerate oligonucleotides weresynthesized using the phosphoramidite method with a Cyclone Plus DNASynthesizer (MilliGen/Biosearch, Burlington, Mass.). Olionucleotideswere synthesized in both the sense and antisense orientations for eachpeptide and used as primers in polymerase chain reactions (PCR). Togenerate the template, DNA complementary to A3.01 polyadenylated RNA wasprepared using avian reverse trtanscriptase (Life Sciences) using themethods disclosed by Sambrook, J., Fritsch, E. F., and Maniatis,T.(1989) Molecular CLoning: A Laboratory Manual, 2nd Ed., Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. DNA amplification wasperformed, using Thermus aquaticus (Taq) polymerase in aPerkin-Elmer/Cetus DNA thermal cycler using the methods disclosed inHeller, R. A., Song, K., Onasch, M. A., Fischer, W. H., Chang, D., andRingold, G. M. (1990), Proc. Natl. Acad. Sci. USA, 87, 6151-6155. Aspecific fragment of approximately 1-kb in length was generated onlywhen oligonucleotides 2S² and 8A² (corresponding to tryptic peptides 2and 8) were used as primers (FIGS. 1 and 4). To confirm the identity ofthis fragment, it was subcloned into pCR.1000 (Invitrogen, San Diego,Calif.). From partial sequence analysis, the sequences corresponding to2S and 8A at the 5' ends of the forward and reverse strandsrespectively, as well as the sequence of oligonucleotide 3S2(corresponds to peptide 3) near the 5' end of the forward strand (seeFIGS. 1 and 4) were detected. This PCR fragment (pcr.2S8A, SEQ ID NO:11) was used to screen an A3.01 cDNA library. Based on subsequentsequence analysis of GMPS.6, the sequence of pcr.2S8A corresponds to thesequence within FIG. 1 starting from residue 781 and ending at residue1952.

Screening of cDNA Library and Sequencing of Positive Clones

A cDNA library, prepared by Stratagene (La Jolla, Calif.), wasconstructed in the Lambda-ZAP II vector system using polyadenylated RNAfrom A3.01 cells. To obtain polyadenylated RNA, total cellular RNA wasisolated according to the method of Chirgwin et al (Chirgwin, J. M.,Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry18, 5294-5299) and polyadenylated RNA was then obtained usingoligo(dT)-cellulose as described by Aviv et al (Aviv, H., and Leder, P.(1972) Proc. Natl. Acad. Sci. USA 69, 1408-1412). The resulting cDNAlibrary prepared by Strategene yielded 9.3×10⁶ primary plaques with thehost bacterial strain SURE (Stratagene, La Jolla, Calif.). Amplificationwas performed on 1×10⁶ plaque forming units (pfu).

The amplified library was screened according to a standard procedureprovided by Stratagene. The PCR fragment pcr.2S8A (SEQ ID NO: 1) wasradiolabeled by random-hexamer priming and it was used as a probe. Atotal of 0.9×10⁶ pfu were screened. After three rounds of screening,eight positive phagemids were excised and rescued by R407 helper phage(Stratagene). Doubled stranded pbluescript (SK⁻) plasmids were isolated,and four of the longest clones were analyzed further by restrictionmapping and found to be overlapping.

The cDNA inserts of the four positive clones were sequenced by themethod of Sanger using fluorescent dye-labeled terminators with a 373ADNA Sequencer (Applied Biosystems). Partial sequence analysis confirmedthat the four clones were overlapping. The complete sequence of bothstrands of the insert of clone 6 (GMPS.6, SEQ ID NO: 1) was determinedand is shown in FIG. 1. Sequence analysis of GMPS.6 revealed an openreading frame of 2079 base pairs (FIG. 1). The first ATG was found at142 bases downstream from the 5' end and a stop codon TAA was found 11bases upstream from the 31 ' end. No poly(A) tail was found on any ofthe clones. The open reading frame yielded a protein of 693 amino acidresidues with a predicted molecular weight of 76,725. The derived aminoacid sequence (SEQ ID NO: 12) of human GMP synthetase is shown inFIG. 1. The predicted size of the enzyme was in good agreement with the75-kDa size indicated by polyacrylamide gel electrophoresis of thepurified A3.01 human GMP synthetase described in Example 1. Thesequences corresponding to the nine tryptic peptides were located withinthe predicted peptide sequence.

Example 3

Construction of Expression Plasmids and Complementation in AT2465 Cells

The insert of GMPS.6 was excised from the pBluescript plasmids obtainedabove and cloned into the Nco I/ Pst I cloning site of the prokaryoticexpression vector, pTRC99A (Pharmacia), to yield vector TRC.6. UsingTRC.6, two expression vectors, NF.6 and ES.6, were constructed to removethe 5'-untranslated sequence of the CDNA. A 201-bp fragment was removedfrom the 5' end of the TRC.6 by digestion with Nco I and Msc I, andreplaced with a 60 base pair-oligonucleotide that had the identicalsequence of residues -1 to 59 except for a G to C change at residue -1(see FIG. 1, residue 1 represents the start site of translation). Thisbase change converted the Bgl I site at the start site of translation toan Nco I site. The 60 base pair-oligonucleotide which has the sequence5'-CATGGC.circle-solid. was directly ligated into the Nco I/Msc Icut vector, and this expression vector was designated NF.6 (NFabbreviates "no fusion"). A second expression vector ES.6 wasconstructed in the same manner except that two stop codons were insertedin the ligated oligonucleotide 39 base pairs downstream from the startsite of translation (ES abbreviates "early stop"). With reference toFIG. 1, the nucleotide sequence from residues 40 to 45 was changed fromGGAGGA.circle-solid. to TGATAA.circle-solid..

To test for complementation of the guanosine requirement, AT2465 cellsthat were transfected with NF.6 or ES.6 were spread on M9 minimal mediaplates with or without guanosine (100 μg/ml). All plates weresupplemented with 0.4% glucose, 0.2% casamino acids, 20 μ/ml IPTG and100 μg/ml of: thiamin, glutamine, histidine, arginine, inosine, biotin,2'-deoxyuridine and ampicillin. Colony formation was examined after16-48 h of growth.

Cells that were not transfected did not produce colonies on either platedue to the absence of ampicillin resistance in the parental AT2465 cells(FIG. 2). Cells that were transfected with either NF.6 or ES.6 producedcolonies in the presence of guanosine. Only cells transfected with NF.6formed colonies in the absence of guanosine. This result showed thatGMPS.6 contained a cDNA that could complement a host E. coli that wasdeficient in GMP synthetase. When this cDNA was mutated in ES.6 by theinsertion of stop codons, it could no longer complement growth in theabsence of guanosine. These data show complementation by the transfectedhuman DNA.

To determine GMP synthetase activity, single colonies were selected andinoculated in LB media containing 100 μg/ml ampicillin. Cultures wereallowed to grow to mid-log phase and then IPTG was added to a finalconcentration of 20 μg/ml. Cells were harvested 3 h after the additionof IPTG.

Determination of Enzyme Activity in Transfected E. Coli Cells

Cells were resuspended in lysis buffer (20 mM Tris-HCl, pH 7.6, 0.5 mMDTT, 0.2 mg/ml lysozyme, 2 mM PMSF, 0.5 mM EDTA, and 10% glycerol) andlysed at 37° C. for 10 min. Genomic DNA was sheared by passing thelysate through a 27 gauge needle three to four times. The cytosolicfraction was separated from the membrane fraction by centrifugation at15,000×g for 15 min. GMP synthetase activity in the cytosol wasdetermined by measuring the formation of [8-¹⁴ C]GMP from [8-¹⁴ C]XMp(50 mCi/mmol, Moravek, Brea, Calif.). The assay mixture contained 75 mMTris-HCl, pH 7.8, 10 mM MgCl₂, 2 mM ATP, 1 mM [¹⁴ C]XMP (10 mCi/mmol), 5mM glutamine and 50 mM DTT. Typically, 5 μl of cytosol was added to 25μl of assay mix, and the reaction was allowed to proceed at 40° C. for15 min. The reaction was stopped by the addition of 6 μl of quenchsolution, which contained 250 mM EDTA and 62.5 mM each of GMP and XMP ascarriers. GMP and XMP were separated by thin layer chromatography asdescribed (Boritzki, T. J., Jackson, R. C., Morris, H. P., and Weber,G.; (1981) Biochim. Biophys. Acta, 658, 102-110). Briefly, the quenchedreaction mixtures (10 μl) were streaked onto 2.5 cm×20 cm strips ofpolyethyleneimine cellulose plates (Macherey-Nagel, Germany). The stripswere developed in 2 M formic acid. The position of GMP was visualized byultraviolet light and marked. The GMP band was excised, and the amountof [¹⁴ C]GMP was determined by liquid scintillation counting. Proteinconcentration was determined by Bradford analysis (Bio-Rad).

The results shown in FIG. 2 demonstrate that GMP synthetase activity waspresent in NF.6 transfected cells and absent in ES.6 transfected cells.Furthermore, the activity was induced by the presence of IPTG. There wasno detectable GMP synthetase activity in untransfected cells. Thesefindings confirm the complementation results and demonstrate that thecloning of a cDNA encoding a functional human GMP synthetase.

Example 4

Expression, Production and Purification of Recombinant Human GMPSynthetase

Lipofection:

A lipid solution consisting of lipofectin (0.067 mg/ml) in HepesBuffered Saline (HBS), (20 mM Hepes, 150 mM NaCl, pH 7.4) and a DNAsolution containing 0.33 ug Autographa californica Nuclear PolyhedrosisVirus DNA (BaculoGold®, PharMingen, San Diego, Calif.) and 3.3 ugtransplacement plasmid (pSYN-GMPS) DNA were prepared.

The transplacement plasmid, pSYN-GMPS, was prepared as follows. Thecoding region of the GMP synthetase cDNA was excised from NF.6 andcloned into the baculovirus expression vector pSynXIV VI⁺ X3 (Wang, X.,Ooi, B. G., and Miller, L. K. (1991) Gene 100, 131-137) to yieldpSyn.GMPS. The vector pSynXIV VI⁺ X3 was linearized at the EcoRI site inthe multiple cloning region downstream from the promoter, and the3'-recessed sequences were filled in using Klenow fragment as describedin Sambrook, J., Fritsch, E. F., and Maniatis, T.(1989) MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. The blunt-ended vector was digested with PstIwhich removed one blunt end. NF.6 was linearized with NcoI which cleavedNF.6 at the start site of translation. Blunt ends were created asdescribed above and the vector was cut with PstI to release the codingregion insert. The insert, which was blunted at the 5'-end and cleavedat the 3'-end with PstI, was cloned into the blunt/PstI cloning site ofthe pSynXIV VI⁺ X3 vector to yield the pSYN-GMPS expression vector.

Plastic flasks (Costar, tissue culture, 25 cm² surface area) were seededwith 3×10⁶ Spodoptera frugiderda (Sf-9) cells (ATCC, Rockville, Md.)that had been adapted to grow in suspension in ExCell-400 medium (JRHScientific). After 1 hour the cells which had attached to the flask werewashed with HBS and three milliliters of lipofection solution consistingof 1 part lipid solution and 1 part DNA solution were added to themonolayer. After 40 minutes at 28° C., three milliliters of ExCell-400medium supplemented with 10% fetal calf serum and gentamicin (50 ug/ml)were added to the lipofection solution in the flask. Thirty minuteslater the lipofection solution was decanted and replaced with freshExCell-400 medium supplemented with 2.5% fetal calf serum. Five dayslater, the culture fluid was collected (5 day harvest) and centrifugedat 2000 rpm for 15 minutes. Fresh Ex-Cell 400 medium supplemented with2.5% FCS was added to the flask and a second harvest is taken 2 dayslater (7 day harvest).

Plaque Purification of Recombinant Baculovirus:

The clarified culture fluid from the transfected cells (either the day 5or day 7 harvest) was serially diluted for 1×10⁻¹ to 1×l0⁻⁶. Six-wellplates (Linbro, tissue culture, 9.6 cm² surface area per well) wereinoculated with 2 ml Sf-9 cell suspension (0.6×10⁶ cells/ mlExCell-400). When the cells attached, 1 hour after seeding, each wellwas inoculated with 0.5 ml of each dilution of the culture fluid induplicate. Viral adsorption continued for 1 hour at 28° C. The plateswere rocked every 15 minutes to facilitate distribution of the virusparticles. The inoculum was then aspirated, and the cell monolayers ineach well overlayed with 2 ml of a solution containing 1 part 2×ExCell-400, 1 part 3% melted Sea-Plaque Agarose, and gentamicin (50ug/ml). The plates were incubated in a humidified environment at 28° C.for 5 days until plaques had developed. A second 2 ml overlay thatcontained 0.1 mg/ml Neutral Red (GIBCO Labs, Grand Island N.Y.) as wellas the components of the first overlay was added to each well. Thefollowing day, plaques were readily visible. When observed under adissecting microscope, polyhedra could be seen at the center of theplaques. Plaques were picked with a P-200 Pippettman® with the volumeadjusted to 100 ul and equipped with a sterile tip. Four well separatedplaques were picked and dispensed into 0.5 ml fresh ExCell-400 medium.The virus from each plaque was repurified three times.

Preparation of Master Seed Virus:

After the third cycle of plaque purification, virus from a single plaquesuspended in 0.5 ml ExCell 400 was inoculated into a well of a 6 wellplate containing 1.2×10⁶ cells. After 1 hour adsorption, 1.5 ml ExCell400 medium was added to each plate. The plates were incubated at 28° C.in a humidified environment for 4-5 days when polyhedra were evident in70-80% of the cells. The culture fluid from each well was clarified bycentrifugation at 1500×g for 10 minutes. Once the culture fluid wastitered, a 100 ml Sf-9 cell culture suspended in a 250 ml plasticErlenmeyer shake flask at a density of 1.2×10⁶ cells/ml was infectedwith the above virus. The ExCell 400 medium was supplemented with 1% BSAor 5% FCS and 4% feed solution. The feed solution consists of 1.25%glutamine, 2.5% glucose, 12.5×lipid concentrate (GIBCO-BRL, GrandIsland, N.Y., cat. #21900-004), and 12.5×yeastolate ultrafiltrate(GIBCO-BRL, Grand Island, N.Y., cat #18200-014). The multiplicity ofinfection was 0.5 plaque forming units per cell. The cells weremaintained in suspension by shaking at 140 rpm in a rotary shaker at 28°C. Four days after infection, the culture fluid was harvested bycentrifugation at 1500×g for 10 minutes. The supernatant was collectedand titered. Aliquots were frozen at -80° C.

Preparation of Working Seed Virus Stock:

An aliquot of master seed virus was thawed and a 100 ml culture of theSf-9 cells growing in ExCell-400 in a 250 ml plastic Erlenmeyer flaskwas infected at a MOI of 0.5. The medium was supplemented with either a1% BSA or 5% FCS and 4% feed solution. The cells were incubated at 28°C. at 140 rpm in a rotary shake flask for four days. The cells were thenpelleted and the culture fluid was utilized as working seed passage 1.The virus was titered and stored at 4° C. until needed. In order toincrease the amount of virus needed for large scale production of humanGMP synthetase working seed passage virus 1 is used to infect fresh Sf-9cells as described above. The virus from this second round of infectionis referred to as working seed passage 2. It is harvested as describedabove and stored at 4° until needed.

Sf-9 cells (1.2×10⁶ /ml) growing in 2 liters of ExCell-400 medium in a 3liter bioreactor (Applikon, Foster City, Calif.) were infected withworking seed passage 1 virus at a multiplicity of infection (MOI) of0.5. The cells were maintained in suspension by rotation of theimpellars at 100-200 rpm. The medium, ExCell-400, was supplemented with0.1% Pluronic F68, 50 ug/ml gentamicin and antifoam AF Emulsion (JRHBiosciences, Lenexa, Kans.) up to 10 ppm. The culture was also fed 80 mlfeed solution at the time of infection. The pH was maintained at 6.3 bytitration with 2N NaOH and the dissolved oxygen was maintained at 30%saturation by sparging with air and adjusting the impellar speed. Afterfour days at 28° C., the cells were pelleted by centrifugation and thesupernatant was titered and stored at 4° C. until needed.

Production of Human GMP Synthetase:

Production of GMPS was carried out in 10 liters of ExCell-400supplemented with 1% Pluronic F68. The cells were infected at a densityof 1.2×10⁶ cells per ml with working seed passage 2 at an MOI of 0.5.The cells were fed with 400 ml feed solution at the time of infection.The culture was maintained at 27° C. and the pH adjusted to 6.3 with 2NNaOH, and 30% saturation dissolved oxygen for three days. The ExCell-400medium was supplemented with gentamicin as described above. The cellswere then harvested by centrifugation and frozen at -80 C. until neededfor purification.

Purification of GMP Synthetase:

Sf-9 cells obtained from above were suspended in lysis buffer (20 mMTris pH 8.0, 0.5 mM EDTA, 0.5 mM DTT, 10% glycerol, 2 ug/ml leupeptin, 2ug/ml pepstatin, 2 ug/ml aprotinin). After mixing for 10 minutes, thecells were transferred to a Dounce (type B) homogenizer and the cellsdisrupted with 20 strokes of the homogenizer. Nuclei and other celldebris were pelleted by centrifugation for 20 minutes at 41,000×g.Ammonium sulfate was added to the supernatant slowly to give a 35%saturation solution. After stirring for 30 minutes, the preparation wassubjected to centrifugation (41,000×g, 20 minutes). The pellet wasdiscarded and additional ammonium sulfate added to the supernatant sothat the final concentration was 60% of saturation. The mixture wasstirred 30 minutes and then subjected to centrifugation (10,000×g, 10minutes). The pellet was resuspended in an equal volume of lysis bufferand transferred to a 6000-8000 mwco dialysis bag. The suspension isdialyzed against 10 volumes of dialysis buffer for 16 hours.

The dialyzed suspension was then centrifuged at 41,000×g for 20 minutes.The supernatant was filtered through a 0.45 μm filter and theconductivity was adjusted to less than 3.6 μMHO with H₂ O. Thepreparation was then loaded onto a Q-Sepharose FF column (Pharmacia) ata linear flow rate of 1.4 cm/min. All subsequent operations were at aflow rate of 20 cm/min. Protein elution was monitored continuously atfollowing the optical density of the eluent at 280 nm. The column wasthen washed with buffer A (20 mM Tris, pH 8, 0.5 mM EDTA, 0.5 mM DTT,10% glycerol) until optical density at 280 nm returned to baseline.Bound proteins were eluted by running a gradient against buffer B (1 MNaCl, 20 mM Tris, pH 8, 0.5 mM EDTA, 0.5 mM DTT, 10% glycerol). Thegradient went to 25% buffer B over 50 minutes. Fractions were assayedfor GMP synthetase activity and those with appreciable activity werepooled.

Ammonium sulfate was added slowly to the pooled fractions from theQ-Sepharose column (1.94 grams per 10 ml). The solution was thenfiltered through a 0.8 μm filter. A Poros PE phenyl ether (PerSeptiveBiosystems, Cambridge, Mass.) column was equilibrated against buffer C(20 mM Tris pH 8, 0.5 mM DTT, 10% glycerol, 19.4% ammonium sulfate) at alinear flow rate of 1.7 cm/min.

The protein was then loaded onto the column which was then washed withthe equilibration buffer until optical density returned to baseline.This and subsequent operations were at 3.4 cm/min. A 0-60% buffer D (20mM Tris pH 8, 0.5 mM DTT, 10% glycerol) gradient was run over a 36minute interval. Fractions were assayed for GMP synthetase activity andthose with appreciable activity were pooled. The protein wasconcentrated to approximately 2 mg/ml using a YM 30 membrane (Amicon)ina stir cell. The protein preparation was mixed with 1 part glycerol andfrozen at -80C. The purified protein had kinetic and biochemicalproperties identical to those described of the purified human GMPsynthetase from A3.01 cells described in Example 1. The specificactivity of the enzyme was 4.4 μmol AMP produced/min/mg.

The above procedure was repeated on several occasions and samples ofrecombinant human GMP synthetase with specific activities in the rangeof 3.0-4.4 μmoles AMP produced/min/mg were routinely obtained.

Example 5

Assay for Inhibitors of Human GMP Synthetase

The source of enzyme was a baculoviral expressed human GMP synthetaseobtained as described above. Alternatively, purified enzyme from A3.01cells obtained as described in Example 1 was also used. Purified humanGMP synthetase enzyme was used in testing. The specific activity of theenzyme used was between 2.5 to 3.0 μmol AMP produced/min/mg. Twoprocedures were used for assaying enzyme activity in the presence of asuspected inhibitor.

Procedure #1: Spectrophotometric-Coupled Assay

The formation of AMP was measured by coupling the GMP synthetasereaction to three auxiliary enzymes: these were AMP kinase, pyruvatekinase and lactate dehydrogenase. In addition to the substrates for GMPsynthetase, also included in the assay mix were phosphoenol pyruvate(substrate for pyruvate kinase) and NADH (substrate for lactatedehydrogenase). Through the activities of these auxiliary enzymes, theformation of AMP was measured by the eventual oxidation of NADH whichwas monitored by the decrease of absorbance at 340 nm. This procedurecould be automated in a high throughput screen. Because this assay is anindirect assay and an active compound can be inhibiting any or all fourenzymes involved in the assay, it is used as a primary screen forquickly identifying potentially active inhibitors.

Procedure #2: Radioactive (Direct) Assay

In this assay, radiolabeled XMP or ATP was used and the formation ofradioactive GMP or AMP was measured directly after separation ofsubstrates and products by thin-layer chromatography. This assay wasused to confirm results from the primary screen. Typically, a suspectedinhibitor was dissolved and diluted in DMSO and the final DMSOconcentration in the assay was 10%.

Assay #1: Spectrophotometric-Coupled Assay For Initial Screen

Materials: Stock solutions are prepared and stored frozen until used.

    ______________________________________                                        Reagent        Concentration                                                  ______________________________________                                        1.  Tris.HCl, pH 7.8                                                                             750 mM                                                       2. MgCl.sub.2 100 mM                                                          3. ATP 400 μM                                                              4. XMP 200 μM                                                              5. glutamine 2000 μM                                                       6. phosphoenolpyruvate 5 mM                                                   7. NADH 1.5 mM                                                                8. KCl 80 mM                                                                  9. dithiothreitol 1M                                                          10. compound or extract of 0 to 200 μM or 50 μg/ml in DMSO (10%                            suspected inhibitor DMSO final)                            11. AMP kinase 1 mg/ml (2000 U/ml)                                            12. pyruvate kinase 10 mg/ml (2000 U/ml)                                      13. lactate dehydrogenase 10 mg/ml (8500 U/ml)                                14. GMP synthetase 20 μg/ml                                              ______________________________________                                    

Method

1. Prepared assay mix by mixing 1 ml each of reagents 1-8, 0.5 ml ofreagent 9 and 0.1375 ml of distilled H₂ O. This is solution A.

2. Pipetted 2.764 ml of solution A into a tube and warm to 40° C.

3. Diluted 50 μl of compounds/extracts (reagent 10) into UV-qualitydisposable cuvettes.

4. Prepared auxiliary enzyme mix by mixing 25 μl of each of reagents 11and 12, and 12.5 μl of reagent 13.

5. Added 20 μl of auxiliary enzyme mix (from step 4) to 2.764 ml of warmassay mix from step 2.

6. Aliquoted 435 μl of the warm assay/auxiliary enzyme mix from step 5into the individual cuvettes from step 3 containing the suspectedinhibitory compounds or extracts.

7. Started reaction by the addition of 15 μl GMP synthetase to thecuvettes from step 6.

The final concentrations of all assay components are shown below.

    ______________________________________                                        Reagent        Final concentration (in assay)                                 ______________________________________                                        1.  Tris.HCl, pH 7.8                                                                             75 mM                                                        2. MgCl.sub.2 10 mM                                                           3. ATP 40 μM                                                               4. XMP 20 μM                                                               5. glutamine 200 μM                                                        6. pyruvate 0.5 mM                                                            7. NADH 0.15 mM                                                               8. KCl 8 mM                                                                   9. dithiothreitol 50 mM                                                       10. compound or extract of 0 to 200 μM or 50 μg/ml in DMSO (10%                            suspected inhibitor DMSO final)                            11. AMP kinase 2.5 μg/ml                                                   12. pyruvate kinase 25 μg/ml                                               13. lactate dehydrogenase 1.25 μg/ml                                       14. GMP synthetase 0.6 μg/ml                                             ______________________________________                                    

The assay was run at 40° C., 10 min, 0.5 ml final volume, monitoringcontinuous change in OD₃₄₀. A negative control was run using 50 μl ofDMSO instead of the suspected inhibitory compound or extract.

Assay #2: Radioactive (Direct) Assay For Confirmation Screen

Materials:

    ______________________________________                                        Reagent          Concentration                                                ______________________________________                                        1.   Tris.HCl, pH 7.8                                                                              750 mM                                                     2. MgCl.sub.2 100 mM                                                          3. ATP 400 μM                                                              4. XMP 2.5 mM                                                                 5. .sup.14 C-XMP 1 mM (50 mCi/mmol)                                           6. glutamine 2 mM                                                             7. dithiothreitol 1M                                                          8. compound or extract of 0 to 200 μM or 500 μg/ml in DMSO                                   suspected inhibitor                                      9. GMP synthetase 2.57 μg/ml                                             ______________________________________                                    

Method

1. Prepared assay mix by mixing 60 μl each of reagents 1, 2, 3, and 6,30 μl of reagent 7, 4.8 μl of reagent 5, 2.9 μl of reagent 4, and 122.3μl of distilled H₂ O. This is solution A.

2. Warmed assay mix (solution A) to 40° C.

3. Diluted 3 μl of compounds/extracts into 0.4 ml Eppendorf tubes.

4. Aliquoted 20 μl of warm assay mix from step 2 into the individualtubes containing the suspected inhibitors.

5. Started the reaction by the addition of 7 μl of GMP synthetase.

6. Stopped the reaction after 10 minutes with 3 μl 500 mM EDTA.

7. Spiked reaction with 3 μl of standards of GMP and XMP (125 mM each).

8. Streaked 5 μl assay mix (twice, total 10 μl) onto PEI cellulose TLCplates (previously cut into 2.5 cm×20 cm).

9. Chromatographed in 2 M formic acid.

10. Visualized GMP band by UV lamp.

11. Excised GMP band and added 4 ml scintillation cocktail (BeckmannReadysafe).

12. Determined ¹⁴ C-GMP formation by liquid scintillation counting.

The assay was run at 40° C. for 10 minutes in a final volume of 30 μl. Anegative control was run using 3 μl of DMSO instead of the suspectedinhibitory compound or extract. Several compounds which inhibited GMPsynthetase by 50% or more under these conditions were identified usingthis screening procedure. The IC₅₀ of some of these compounds is shownbelow.

    ______________________________________                                               Compound                                                                              IC.sub.50                                                      ______________________________________                                               A       30           μM                                               B 3 μM                                                                     C 0.5 μM                                                                   D 7 μM                                                                     E 3 μM                                                                     F 10 μM                                                                  ______________________________________                                    

Example 6

Generation of Anti-GMP Synthetase Antibody

An ˜300 base pair Hind III/Sac I fragment from the middle of the codingregion of clone 6 (GMPS.6) obtained in Example 2 (see FIG. 4) wassubcloned into the bacterial expression vector pGEX-2T (Pharmacia) andexpressed as a glutathione transferase fusion protein in SURE bacteria.This fusion protein was isolated using a glutathione sepharose columnaccording to Pharmacia instructions. A one liter culture yielded 1.5 mgof protein, which was used to immunize rabbits according to an approvedprotocol. Rabbit sera were collected and the immunoglobulin fraction wasisolated. Purified human GMP synthetase was used in immunoblot analysisto test for the presence of antibodies against human GMP synthetase. Inthis fashion polyclonal rabbit antibodies were obtained.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A method for purifying human GMP synthetaseproduced by host cells expressing a polynucleotide comprisingnucleotides 148 to 2226, inclusive of SEQ ID NO:1 comprising,(a)homogenizing said host cells and isolating the cytosolic component, (b)fractionating said cytosolic component to its protein fraction bydifferential salt fractionation and (c) desalting and subjecting saidprotein fraction to anion exchange and hydrophobic interactionchromatographic purification.
 2. An isolated polypeptide encoded by apolynucleotide comprising nucleic acids 1-2237 of SEQ ID NO:1.
 3. Anisolated polypeptide encoded by a polynucleotide comprising nucleicacids 928-2100, inclusive, of SEQ ID NO: 1.